Drug resistant HIV-1 variants that arise under the selective pressures of antiretroviral treatment exhibit not only reduced susceptibility to antiretroviral compounds, but also impaired fitness relative to the wild-type virus. Compensatory amino acid substitutions in the protease (PR) and reverse transcriptase (RT) regions, as well as gag cleavage sites can restore viral replicative potential to a varying degree despite the presence of drug resistance conferring mutations. In fact, we have identified viral isolates from newly infected individuals that exhibit resistance to at least two classes of drugs yet replicate to high levels in vitro and in vivo. Interestingly, the introduction of PR-RT regions that encode the multi-drug resistance (MDR) phenotype into a wild-type genetic background causes a pronounced replication defect that is only partially rescued by complementation with the isogeneic gag. The marked discrepancy in fitness between viral isolates and the chimeras encoding gag/PR/RT suggests that regions 3' to those that confer drug resistance play a crucial role in overcoming defects present in viable MDR viruses. In this experimental proposal, we aim to identify the adaptive changes and the molecular mechanisms that are essential for restoration of infectivity in these MDR variants. To date, replication-competent molecular clones with MDR phenotype have not been generated. These experiments will assess the fitness of reconstructed MDR molecular clones derived from MDR isolates that are well adapted to replicate to high levels in the absence of drugs. We will analyze the relative contribution of different regions of the MDR viral genome to fitness in both wild-type and MDR HIV-1 genetic backgrounds. We will also attempt to define the amino acid substitutions that are required for restored fitness in a MDR context using site-directed mutagenesis. In addition, we will determine whether the MDR-associated replication defects map to early or late stages of the viral life cycle, and assess whether viral protein expression and processing is altered. Finally, we will monitor the genetic stability of reconstructed replication-competent MDR clones encoding isogeneic or wild-type PR-RT in cell culture. These studies will help elucidate novel viral mechanisms that confer high replication capacity despite the presence of a MDR phenotype. In this way we will gain insight into the special challenges posed by compensatory adaptations displayed by some HIV-1 strains in the face of drug treatment.